1. Field of the Invention
The present invention relates to the isolation, purification and characterization of derivatives of 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (nordihydroquaiaretic acid, NDGA). The derivatives were isolated from leaf and flower extracts of the creosote bush (Larrea tridentata, Zygophyllaceae) and together with NDGA can be used to suppress Tat transactivation in lentiviruses, including the HIV virus.
2. Description of the Related Art
Tat is a transactivator of human immunodeficiency virus (HIV) gene expression and is one of the two or more necessary viral regulatory factors (Tat and Rev) for HIV gene expression. Tat acts by binding to the TAR RNA element and activating transcription from the long terminal repeat (LTR) promoter.
The Tat protein stabilizes elongation of transcription and has also been shown to be involved in transcription initiation. Previous studies have shown that Tat mediates reduction of antibody-dependent T cell proliferation, contributing substantially to the failure of the immune response. Tat also directly stimulates Kaposi""s cell growth.
Since Tat has no apparent cellular homologs, this strong positive regulator has become an attractive target for the development of anti-AIDS drugs (see FIG. 1). In contrast to currently available HIV reverse transcriptase inhibitors (AZT, DDI) or potential protease inhibitors that prevent new rounds of infection, an inhibitor which suppresses viral gene Tat regulated expression of integrated proviral DNA will arrest the virus at an early stage (Hsu et al., Science 254:1799-1802, 1991).
Efforts aimed at the elucidation of factors which control gene expression at transcriptional and post-transcriptional levels in host eukaryotes have recently made possible quantitative assessment of Tat function (Sim, Ann. N.Y. Acad. Sci. 616: 64-70, 1990, the entire contents of which are hereby incorporated by reference and relied upon). To screen for inhibitors for Tat regulated transactivation (Tat-TRS), the secreted placental alkaline phosphatase (SEAP) reporter gene is put under the control of HIV-1 LTR promoter in the plasmid pBC12/HIV/SEAP. The Tat-coded activity is supplied by a second plasmid construct pBC12/CMV/t2. Transient cotransfection of COS cells with these two plasmids leads to secretion of alkaline phosphatase into the culture medium which is analyzed by a simple calorimetric assay (Berger et al., Gene 66: 1-10, 1988, the entire contents of which are hereby incorporated by reference and relied upon). The SEAP assay, therefore, provides an indirect determination of Tat transactivation. An inhibitor should cause reduction of the SEAP activity which is due to inhibition of the expression of the SEAP mRNA via transactivation of the HIV-1 LTR promoter by Tat protein (Tat-TRS).
In the present application, we disclose Tat-TRS inhibitory activity of the desert plant Larrea tridentata. Among several plant extracts prepared from rain forest and desert medicinal plants used in traditional medicinal against viral affections, only the total extract from the leaves and flowers of the creosote bush (Larrea tridentata) showed Tat-TRS inhibitory activity. This extract also inhibits HIV cytopathic effects on human lymphoblastoid cells chronically infected with the virus as assessed by the newly developed soluble-formazan assay (Weislow et al., JNCI 81: 577-586, 1989, the entire contents of which are hereby incorporated by reference and relied upon).
The present invention discloses compounds of the structural formula: 
wherein R1, R2, R3 and R4 are each selected from the group consisting of HOxe2x80x94, CH3Oxe2x80x94 and CH3(Cxe2x95x90O)Oxe2x80x94, provided that R1, R2, R3 and R4 are not each HOxe2x80x94 simultaneously.
Each compound was isolated from leaf-flower extracts of the creosote bush Larrea tridentata and is a derivative of 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (nordihydroquaiaretic acid, NDGA).
In addition, NDGA and each derivative can be used to suppress Pat transactivation of a lentivirus, including the HIV virus, in a cell by administering NDGA or a derivative thereof to the cell.